The overall objective of this project is to attempt to correlate nucleic acid modifications, by simple alkylating carcinogens, with nucleic acid function and structure in vivo, using, in part, in vitro model experiments. Although DNA in mammalian cells does not differ from DNA in bacteria in its chemical reactivity, there is a quantitative difference in repair. Repair, or lack of repair, may be the critical event determining whether a promutagenic modified base in DNA can initiate carcinogenesis by somatic mutation. The carcinogen we now use, ethylnitrosourea, can ethylate all oxygens and nitrogens in the DNA of a variety of tissues from treated rats. Most of the chemical derivatives formed have been shown to cause misincorporation in transcription in a single system, but partial repair of all has been demonstrated inrat liver and human cells. There are three aspects of initiation by alkylating carcinogens that can be separated and individually studied in this laboratory. These are: (1) effect of variously modified bases on fidelity in transcription by both DNA and RNA-dependent RNA polymerases; (2) effect of misincorporation in transcription on the physical structure of the synthesized doublestranded polynucleotide; and (3) identification and characterization of alkyl repair enzymes existing in mammalian systems. We will prepare single- and doublestranded ribo- and deoxyribo-polynucleotides, each containing a single one of various modified nucleosides, to study their transcription with procaryotic and eucaryotic polymerases. Misincorporation in transcription (i.e. mutagenesis) will be assessed by nearest neighbor analyses. The products of transcription will be studied in terms of their physical structure as another measure of the ability of a modified nucleoside to substitute for a normal one. The same type of polymers but with highly labeled modified bases will be used as substrates to search for mammalian enzymes which can remove/repair/dealkylate N- and O- ethyl nucleotides an dethyl triesters. The specificity of such enzymes toward ethyl groups, compared to methyl groups, may give additional insight into the higher carcinogenicity of ethylating agents.